During division, cells must partition replicated chromosomes equally to each daughter cell. If this process goes awry, cells accumulate extra chromosomes or partial chromosomes, leading to an aberrant state of aneuploidy, a hallmark of cancer. Chromosome segregation is driven by a cellular machine called the mitotic spindle, which emanates microtubules that attach to chromosomes. Multi-component complexes called kinetochores link chromosomes to microtubules and serve as regulatory sites that sense when chromosomes are properly oriented along the spindle. Kinetochores are essential for maintaining the fidelity of division, making them appealing targets for cancer therapeutics. Determining the function of individual kinetochore components is crucial to understanding how the entire kinetochore operates as a whole. My overall goal is to reconstitute the kinetochore in vitro, and I will begin by building kinetochore complexes from the microtubule-binding end inward. With this approach I will use single molecule assays to define how the central components organize and influence the function of the outer kinetochore complexes. By systematically reconstructing each kinetochore component in vitro, we can examine individual activities and interactions between the complexes to fully appreciate their role within a larger structure. Understanding how the kinetochore operates at the molecular level is key to identifying mitotic targets that will aid the development of new and better cancer treatments.